Acid-producing bacteria are used extensively in the manufacture of fermented meat products and silage, as well as in the manufacture of yogurt, cheese, and the like. Lactic acid-producing bacteria are particularly useful in these manufacturing processes. Typical methods for storing these bacteria involve freezing, freeze-drying, and/or microencapsulation.
Freezing has been widely used for storage of microorganisms, and extensive research has been conducted to determine the optimum conditions for maintenance of maximum activity in frozen cultures. See, for instance, "Frozen Starters from Internal pH-Control-Grown Cultures", Technical Paper No. 6427, Oregon Agricultural Experiment Station, Journal of Dairy Science, Vol. 67, No. 1, (1984), Thunnel and Sandine.
Additionally, for over 20 years, freeze-drying (lyophilization) has been employed in order to preserve lactic acid-producing bacteria. See, for instance, Morichi, T., "Preservation of Lactic Acid Bacteria by Freeze-drying", JARQ, No. 3, Pages 170-176 (1974). Also, Suzuki in U.S. Pat. No. 4,217,419 (1980), discloses lyophilization of a suspension of Lactobacillus containing glucose and sodium alginate.
However, there are many problems accompanying these processes. As the freezing takes place, it kills some of the cells. Likewise, when it is decided to rehydrate the stored material, the rehydration process kills additional cells. Thus, carriers are employed to protect the culture during the freezing and/or freeze-drying process. Typical carriers are fermentable carbohydrates, such as glucose, lactose or sucrose. The presence of the fermentable carbohydrates, however, will cause the microorganism to produce acid, resulting in injury and/or death to some of the bacterial cells due to the reduction in pH to a level below that which is optimal.
Encapsulating techniques and encapsulated products for the controlled release of product as a function of the destruction of the encapsulating material over a period of time are also well established in the prior art as methods to preserve microorganisms. By protecting microorganisms from the outside environment, the capsule enables the microorganisms to continue to function. For instance, Lim and Moss, "Microencapsulation of Living Cells and Tissues", Journal of Pharmaceutical Sciences, Vol. 70, No. 4, Pages 351-354 (April, 1981), disclose a microencapsulation procedure for viable cells using calcium chloride to gel cells suspended in sodium alginate droplets.
More recent studies relate to enhancing preservation of microorganisms without using the well established freezing, freeze-drying, or microencapsulation techniques.
For instance, use of a salt to preserve microorganisms without freezing is shown in U.S. Pat. No. 4,308,287 (Kahn and Eapen). This patent discloses a method to enhance preservation of yogurt (which contains acid producing bacteria) by using quinine salts.
Another item of literature disclosing the use of a salt for preservation is Smith, Benedict, and Palumbo, "Protection Against Heat Injury in Staphylococcus aureus by Solute", Journal of Food Protection, Vol. 45, No. 1, Pages 54-58, (January, 1982). This article discloses the use of salts such as sodium citrate, KCl, NaNO.sub.3, Na.sub.2 SO.sub.4, Na.sub.2 HPO.sub.4, NH.sub.4 Cl, CaCl.sub.2, and LiCl for protection against heat-injury with a concomitant decrease in the number of injured S. aureus cells over a 90-minute heating-time interval at 49.degree. C.
Storage at 4.degree. C. of suspensions of Streptococcus lactis E and S. cremoris using lactose as the carrier is disclosed in Cowell, Koburger, and Weese, "Storage of Lactic Streptococci. I. Effect of pH on Survival and Endogeneous Metabolism in Phosphate Buffer", West Virginia University Agricultural Experiment Station, Paper No. 859 Pages 365-369, (January, 1966). However, as can be seen from FIG. 2 on Page 366 of this reference, a decrease in activity or viability starts around 5 days and becomes substantial by 10 days, even when the lactose-containing suspension is buffered at the optimum pH of 8.5. Thus, Cowell et al do not show how to maintain constant viability for storage of acid-producing bacteria at a temperature above freezing. Rather, they show that buffering at a pH of 8.5 achieves a reduction in loss of viability for storage of lactose-containing suspensions of bacteria at 4.degree. C.